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Control of Programmed Cyclin Destruction in a Cell-free System

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Control of Programmed Cyclin Destruction in a Cell-free System Powered By Docstoc
					Published November 1, 1989


    Control of Programmed Cyclin Destruction in a Cell-free System
    Francis C. Luca and Joan V. R u d e r m a n
    Department of Anatomy and Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; and Departments of Cell Biology
    and Zoology, Duke University, Durham, North Carolina 27706




    Abstract. To ask what controls the periodic accumu-                  Mg 2+. By combining lysates from different cell cycle
    lation and destruction of the mitotic cyclins across the             stages, we show that (a) interphase lysates do not con-
    cell cycle, we have developed a cell-free system from                tain a dominant inhibitor of cyclin destruction and (b)
    clam embryos that reproduces several aspects of cyclin               the timing of cyclin destruction is determined by the
    behavior. One or more rounds of cyclin proteolysis                   cell cycle stage of the cytoplasm rather than the cell
    and resynthesis occur in vitro, and the destruction of               cycle stage of the substrate cyclins themselves. Among
    the cyclins is highly specific. The onset, duration, and             a large variety of agents tested, only a few affect cyclin
    extent of cyclin destruction and the appropriately stag-             destruction. Tosyl-lysine chlormethyl ketone (TLCK, a




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    gered disappearance of cyclin A and cyclin B are cor-                protease inhibitor), 6-dimethylaminopurine (6-DMAP,
    rectly regulated during the first cycle in the cell-free             a kinase inhibitor), certain sulfhydryl-blocking agents,
    system. Just as in intact cells, lysates made from early             ZnC12 and EDTA (but not EGTA) completely block
    interphase cells require further protein synthesis to                cyclin destruction in vitro. Addition of 1 mM Ca 2+ to
    reach the cyclin destruction point, and lysates made                 the cell-free system has no effect on cyclin stability,
    from later stages do not. Using the cell-free system we              but 5 mM Ca 2+ leads to the rapid destruction of cy-
    show that cyclin disappearance requires ATP and                      clins and a small number of other proteins.



        s rapidly dividing embryos of marine invertebrates, new-           A regulatory role for the cyclins was first inferred from the

    I    ly synthesized cyclins accumulate steadily across the
         cell cycle and then abruptly disappear near the end of
    mitosis (reviewed by Swenson et al., 1989). Several different
                                                                         observation that cyclin levels oscillated in a way that could
                                                                         explain why newly made proteins are needed to enter M
                                                                         phase in each cell cycle (Evans et al., 1983). Several types
    experimental approaches have established that the disappear-         of evidence now show that newly synthesized cyclins are re-
    ance of cyclin near the end of each cycle is due to a burst of       quired for the generation of active maturation or M phase-
    rapid and selective proteolysis (Evans et al., 1983; Swenson         promoting factor (MPF) t and entry into M phase. First,
    et al., 1986; Westendorf et al., 1989; Murray and Kirschner,         when introduced into frog oocytes that are naturally arrested
    1989). In clam embryos, there are two prominently synthe-            in meiotic prophase, cyclins induce entry into meiotic M
    sized cyclins, cyclin A and cyclin B. While the levels of both       phase (Swenson et al., 1986; Pines and Hunt, 1987; Westen-
    oscillate across the cell cycle and both can act as M phase          dorf et al., 1989). Conversely, depleting activated egg lysates
    inducers, the two are distinguished by their amino acid se-          of endogenous cyclin mRNAs prevents nuclei from entering
    quences, their differential patterns of expression during oo-        mitotic M phase (Minshull et al., 1989). Second, newly syn-
    genesis and meiosis, and their slightly offset kinetics of de-       thesized cyclin joins with a preexisting component of active
    struction (Swenson et al., 1986; Westendorf et al., 1989;            MPF (Draetta et al., 1989), a 34-kD protein kinase origi-
    Hunt, T., and J. Ruderman, unpublished). In normal meiotic           nally identified as the gene product of the fission yeast cdc2/
    and mitotic cycles, the proteolysis of cyclin A begins in late       budding yeast cdc28 gene (Gautier et al., 1988; Dunphy et
    metaphase and is completed within ,',,5-6 min; the drop in           al., 1988) and probably a small number of other proteins
    cyclin B begins a few minutes after that of cyclin A and is          (Lohka et al., 1988; Westendorf et al., 1989) to generate a
    similarly completed within a 5-6 min window. A and B cy-             complex that resembles active MPF in several key ways.
    clins have been identified in Drosophila, where they show            Whereas the relative level of p34'~v2s protein kinase remains
    dramatically different patterns of transcription in somatic          constant across the cell cycle, its activity changes markedly.
    and germ cell lineages (Lehner and O'Farrell, 1989; Whit-            When assayed for activity towards histone HI, a highly pre-
    field et al., 1989), and in Xenopusand yeast, where they have        ferred substrate, both MPF kinase activity and p34~2~28ki-
    been designated as A or B type on the basis of sequence ho-
    mologies with clam cyclins (Reed et al., 1988; Solomon et            1. Abbreviations used in this paper: MPE M phase-promoting factor;
    al., 1988; Goebl and Byers, 1988; Nash et al., 1988; Hagan           NEM, n-ethyrnaleimide; pHMB, p-hydroxymercuribenzoic acid; 6-DMAP,
    et al., 1988; Minshull et al., 1989).                                6-dimethylaminopurine; TLCK, tosyl-lysine chloromethyl ketone.



     © The Rockefeller University Press, 0021-9525/89/11/1895/15 $2.00
     The Journal of Cell Biology, Volume 109, November 1989 1895-1909     1895
Published November 1, 1989


    nase activity are low in interphase, rise markedly at M phase,                 clinical centrifuge and rinsed two to three times in cold (0°C) calcium-free
    and drop abruptly just around the time when the cyclins are                    sea water and once in cold buffer T (300 mM glycine, 120 mM K-gluconate,
                                                                                   100 mM taurine, 100 mM Hepes, 40 mM NaCI, 2,5 mM MgCI2, adjusted
    destroyed (Sano, 1985; Meijer et al., 1987; Draetta and                        to pH 7.2 with KOH). After the third wash, all buffer was aspirated from
    Beach, 1988; Arion et al., 1988; Draetta et al., 1989; Labb6                   the cell pellet and the cells were homogenized with a stainless steel Dounce
    et al., 1989; Murray and Kirschner, 1989). Third, in yeast                     homogenizer (Wheaton Instruments Division, Milville, N J). The homoge-
    there is compelling genetic and biochemical evidence for a                     nate was centrifuged twice at 12,000 g for 15 min. This step was essential:
    physical association between cyclins and p34 °~c2/28that is re-                cyclin destruction was always seen in 12,000 g supematants but never in un-
                                                                                   fractionated homogenates. Aliquots of the final supernatant were frozen in
    quired for cells to enter mitosis (Reed et al., 1988; Booher                   liquid nitrogen and stored at -80°C. Each microliter of lysate represented
    and Beach, 1988; Solomon et al., 1988; Goebl and Byers,                        •1.5 x 104 homogenized cells.
    1988; Nash et al., 1988; Booher et al., 1989).                                    Early interphase lysates were made from cells collected ,,o5-10 min after
       Our recent work suggests that active MPF is generated                       the second polar bodies had formed (,x,60-65 min after fertilization); at this
                                                                                   time pronuclear envelopes were not detectable by phase-contrast micros-
    when p34 ~¢2/2sis complexed with rising levels of cyclin, and                  copy. Mid-interphase lysates were prepared from cells that had progressed
    MPF activity is lost as a consequence of the proteolytic de-                   further to form well demarcated pronuclear membranes that were easily
    struction of cyclins near the end of M phase (Draetta et al.,                  seen by phase-contrast microscopy (*70-75 min after fertilization). To
    1989; Westendorf et al., 1989). The experiments of Murray                      make lysates from cells arrested in interphase of the second mitotic cell cy-
                                                                                   cle, 100 #M emetine (Sigma Chemical Co., St. Louis, MO; Lodish et al.,
    and Kirschner (Murray and Kirschner, 1989; Murray et al.,
                                                                                   1971) was added to embryos at the onset of first mitosis. This concentration
    1989) directly confirm this, although these authors argue that                 inhibits protein synthesis by >99%. Embryos passed through first mitosis
    cyclins are catalytic activators of MPF rather than essential                  on schedule and entered second interphase but never entered second mito-
    components. Because the periodic rise and fall in cyclin lev-                  sis. After control embryos had reached the four-cell stage, emetine-arrested,
    els is controlled by brief, periodic bursts of highly selective                two-cell embryos were collected and homogenized.
    proteolysis near the end of M phase, it is important to under-
    stand what regulates cyclin destruction. Is cyclin protease                    In Vitro Cyclin Destruction Assay
    constitutively active throughout the cell cycle, with cyclin de-               [3sS]Methionine-labeled lysates were thawed on ice, diluted 1:1 with buffer




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    struction being regulated by periodic changes in the accessi-                  T, and incubated at 18"C. At various intervals after the start of the incuba-
                                                                                   tion in vitro (typically 2, 5, 10, 20, 30, and 60 rain; 2, and 5 h), 5-fd aliquots
    bility of the substrate cyclins? Or, instead, is cyclin protease               were taken, added to 95 ~l SDS sample buffer (Laemmli, 1970) containing
    inactive across most of the cell cycle then briefly activated                  5% ~-mercaptoethanol, and boiled for 2-5 rain. Samples were electropho-
    near the end of each M phase? Is there one cyclin protease                     resed on 15 % polyacrylamide gels containing SDS (Anderson et al., 1973),
    or two, one for cyclin A and one for cyclin B? To answer some                  processed for autoradiography and exposed to Kodak XAR-5 film (Eastman
    of these questions, we turned to the use of a cell-free system,                Kodak, Rochester, NY) for 8-24 h.
    an approach that has been very productive for studying the
    regulation of several other aspects of cell cycle control (re-                 Protease lnhibitors
    viewed by Lohka and Mailer, 1987; see also Burke and Ger-
                                                                                   Stocks of PMSF (Sigma Chemical Co.), calpain inhibitor l, calpain inhibi-
    ace, 1986; Dessev et al., 1989). We have prepared a cell-free                  tor II and E64 (Boehringer Mannheim Biochemicals, Indianapolis, IN)
    system from clam embryos that reproduces several features                      were dissolved in DMSO and used immediately. E64d, a gift from Dr. K.
    of in vivo cyclin behavior, including the programmed rise                      Imahori (Mitsubishi-Kasai Institute, Tokyo), was dissolved in DMSO.
    and fall of the cyclins and the temporally offset destruction                  Stocks of tosyl-lysine chloromethyl ketone (TLCK; Sigma Chemical Co.),
    of cyclin A and B. We show that interphase cells do not con-                   leupeptin and chymostatin (Sigma Chemical Co.) were dissolved in distilled
                                                                                   water and stored at - 2 0 ° C for up to 1 mo. Aprotinin (Sigma Chemical Co.)
    rain a dominant inhibitor of cyclin destruction, and that the                  and soybean trypsin inhibitor (SBTI; Sigma Chemical Co.) were dissolved
    timing of cyclin destruction is determined by the cell cycle                   in water and stored at - 2 0 ° C for not more than 6 mo. Heroin (Sigma Chem-
    stage of the cytoplasm rather than the cell cycle stage of the                 ical Co.) was dissolved in buffer T and stored at -20°C. 6-dimethylamino-
    substrate cyclins. Of the numerous protease inhibitors and                     purine (6-DMAP, Sigma), ~2-macroglobulin and (4-amidinophenyl) me-
                                                                                   thanesulfonyl fluoride (APMSF) (both from Boehringer Mannheim
    other agents tested in the cell-free system, all equally affect,               Biochemicals) were made fresh in either distilled water or buffer T. Stocks
    or fail to affect, the destruction of cyclin A and B. This result              of p-hydroxymercuribenzoic acid (pHMB, Sigma Chemical Co.), n-ethyl-
    suggests that if there are A and B type cyclin proteases, they                 maleimide (NEM; Sigma Chemical Co.), iodoacetamide (Sigma Chemical
    are remarkably similar.                                                        Co.), and dithiothreitol (DTT; Bochringer Mannheim Biochemicals) were
                                                                                   dissolved in buffer T just before use. Stocks of CaCI2, CuC12, LiCI, ZnCI2,
                                                                                   NaF, EDTA, and EGTA were made in buffer T and stored at 4°C. Colcemid
    Materials and Methods                                                          and calcium ionophore A23187 (both from Sigma Chemical Co.) were dis-
                                                                                   solved in DMSO and stored in the dark at -20°C.
    Fertilization and Labeling of Clam Embryos
                                                                                   ATP-regeneratingand ATP-depletion Systems
    Full-grown Spisula solidissima oocytes and sperm were collected and fertil-
    ized as described by Westendorf et al. (1989). Oocytes were suspended at       When indicated, lysates contained an ATP-regenerating system consisting
    a known concentration, typically 20,000-40,000/mi in sterile sea water, fer-   of 50 /zg/mi creatine kinase and 10 mM creatine phosphate both from
    tilized, and cultured at 18"C in the presence of 1 #g/ml Hoechst 33342         (Sigma Chemical Co.). ATP (Sigma Chemical Co.) was dissolved in buffer
    (Calbiochem-Behring Corp., La Jolla, CA). Afar entry into meiosis I at         T, pH 7.2, before adding to the lysates. ATPT-S (Boehringer Mannheim Bio-
    11-12 rain after fertilization, embryos were concentrated to 200,000/ml and    chemicals) was dissolved in buffer T and then added immediately to an
    cultured further at 18°C. Progress through the meiotic and mitotic cell cy-    equal volume of lysate. To deplete the endogenous ATE hexokinase (Sigma
    cles was followed by monitoring the state of H33342-stained chromosomes        Chemical Co.) and glucose were added to final concentrations of 100 U/ml
    in living cells by fluorescence microscopy. If embryos were to be radiola-     and 10 mM, respectively.
    beled, 25/~Ci 35S-labeled Trans label (1,000-1,200 Ci/mmol; ICN Radio-
    chemicals, Irvine, CA) was added per ml of cell culture upon the completion    Dilute Lysates as Substrates for the Cyclin
    of second meiosis, as marked by the appearance of second polar bodies.         Destruction Assay
    Preparation of the Cell-free System                                            [35S]Methionine-labeled lysates were diluted eightfold with buffer T, usu-
                                                                                   ally containing a final concentration of 200 t~M emetine, so that the ratio
     1 × 107 embryos at the stages indicated below were pelleted rapidly in a      of lysate volume/total volume was 0.125. 1 vol of dilute, radiolabeled lysate



    The Journal of Cell Biology, Volume 109, 1989                                  1896
Published November 1, 1989

   was then added to one volume of an unlabeled concentrated test lysate. This      few minutes later. The drop in cyclin B immediately precedes
   brought the ratio of radiolabeled lysateltotal volume to 0.063 and that of the   and parallels the time course of the metaphase-anaphase
   test lysate to the standard ratio of 0.50. At each time point, 5-t,l aliquots
   were taken, added to 45/~1 SDS sample buffer and analyzed by gel elec-           transition. Cyclin A completely disappears at the end of each
   trophoresis followed by autoradiography. When incubated with buffer alone        mitosis, but cyclin B levels drop by only 85-90% (Swenson
   in the ratio of 0.063 lysate/total volume, none of the lysates showed any de-    et al., 1986; Westendorf et al., 1989; Hunt, T., and J. Ruder-
   tectable cyclin proteolysis over the 5-h period assayed.                         man, manuscript in preparation).

   lmmunoprecipitation                                                              Cyclin Destruction Occurs In Vitro
   At each time point across the cyclin destruction assay, 10 tzl of the concen-    Lysates were prepared and frozen from embryos cultured at
   trated [3SS]methionine-labeled lysate (representing '~150,000 cells) was         18°C, labeled with [3sS]methionine during interphase of the
   diluted 50-fold in buffer T. This was incubated for at least 4 h at 4°C with     first mitotic cycle and collected at the onset of first mitosis
   5-10 ~1 preimmune or immune rabbit polyclonal sera containing antibodies         (82-84 rain after fertilization). To follow the fate of the la-
   directed against about three quarters of the length of cyclin A or cyclin B
   proteins 0Vestendorf et al., 1989). Immune complexes were then precipi-          beled cyclins in vitro, an aliquot was thawed, diluted with
   tated by adding 50 ~1 of a 1:1 slurry of protein A-Scpharose CL4B beads          one volume of buffer, and incubated at 18°C. Samples were
   (Pharmacia Fine Chemicals, Piscataway, NJ) and buffer, and incubaled for         taken at various times after the start of the incubation, typi-
   another 4 h at 4"C. After the final incubation, beads were washed three          cally 2, 5, 10, 20, 30, and 60 rain and 2 and 5 h, and analyzed
   times in buffer T containing 0.2% Tween 20 and three times in TBS (50
   mM Tris, 150 mM NaCI, pH 7.4). Immune complexes were eluted by boil-
                                                                                    by SDS gel electrophoresis followed by autoradiography.
   ing in SDS sample buffer containing 5% j3-mercaptoethanol for 2-5 min,              In the experiment shown in Fig. 2, the protein synthesis
   and analyzed by gel electrophoresis followed by autoradiography.                 inhibitor emetine was added to the lysate at the start of the
                                                                                    incubation (t = 0 min) so that the destruction of prelabeled
    In Vitro Transcription and Translation                                          cyclins would not be masked by the appearance of newly syn-
    SP6-cyclin A and eyclin B mRNAs were transcribed in vitro as described
                                                                                    thesized ones. Most proteins, such as ribonucleotide reduc-
    previously (Swenson et al., 1986; Westendorf et al., 1989). Total RNA was       tase (RR), were stable in vitro for at least 5 h. In contrast,




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    isolated from emetine-arrested two-cell embryo lysates as described by          cyclin levels remained high for several minutes and then
    Rosenthal el al. (1980). RNAs were translated in a rabbit retieulocyte lysate   dropped precipitously, with the disappearance of cyclin A
    (Promega Biotec, Madison, WI) in the presence of [3~S]methionine (175           preceding that of cyclin B. Indeed, in almost all lysates pre-
    Ci/mM final concentration from a 1,444 Ci/mM stock; Amersham Corp.,
    Arlington Heights, IL).                                                         pared from early M phase cells, the destruction of cyclin A
                                                                                    began 5-10 min after the start of the in vitro incubation, a
                                                                                    little later than would have occurred in the intact embryo.
    Mixed Lysate Experiments: Cyclins Made in                                       Once initiated, most cyclin A disappeared within a period of
    Reticulocyte Lysate as Substrates in Clam                                       5 min and virtually all of it was gone by the next time point.
    Embryo Lysates                                                                  Thus, for cyclin A, the onset, duration, and extent of destruc-
    Various amounts of reticulocyte lysate containing SP6-cyclin mRNA trans-        tion in vitro closely resembled that seen in vivo. The be-
    lation products (ranging from one-half to one-sixty-fourth the total volume)    havior of cyclin B was also reproduced in vitro, but less well.
    were added to previously characterized [3SS]methionine-labeled or -unla-        In the lysate shown in Fig. 2, the drop in cyclin B began "o10
    beled M phase lysates in the presence of 100 t~M emetine and incubated
    at 18°C. Aliquots were taken at stated intervals and analyzed as above.         min and had ceased by the next time point 10 min later. These
    Added reticulocyte lysate, with or without eyclin translation products, did     times are slightly delayed compared with the times routinely
    not affect the destruction of endogenous cyclins contained within the clam      seen with intact cells. These sorts of delays and extensions
    lysates.                                                                        in cyclin B proteolysis were typical of most lysates. Curi-
                                                                                    ously, the details of cyclin B destruction, whereas identical
                                                                                    in all aliquots of an individual lysate, varied among different
   Results                                                                          lysates. In general, the onset of cyclin B destruction was
                                                                                    delayed by several minutes, the duration was longer and the
   Timing of the First Mitotic Cell Cycle
                                                                                    final extent of destruction was less.
   In Spisula, full-grown oocytes are arrested at the G2/M bor-                        No obvious intermediates of cyclin destruction were seen
    der of meiosis I. Upon fertilization, the embryos go through                    during the in vitro destruction assay. This indicates that,
    the two meiotic divisions and then begin a series of rapid mi-                  whatever pathway is used to destroy the cyclins, it operates
    totic divisions (Fig. 1). Embryos from an individual batch of                   rapidly and efficiently in the cell-free system. Most impor-
    oocytes goes through the early cell cycles with considerable                    tantly, even after multiple rounds of freezing and thawing, ly-
    synchrony. For different batches, the exact times vary by a                     sates retained the ability to selectively destroy the cyclins on
    few minutes. When cultured at 18°C, embryos generally pass                      schedule. This feature allowed us to check the basic charac-
    through the metaphase-anaphase transition of meiosis II and                     teristics on an individual lysate, and then go on to assay other
    put out the second polar body at 55-60 min after fertilization.                 aliquots of the same lysate under a variety of experimental
    They enter interphase of the first mitotic cell cycle at 60-65                  conditions.
    min, as marked by chromosome decondensation and forma-
    tion of distinct pronuclear envelopes. Entry into first mitotic
    M phase, signaled by chromosome condensation and nuclear                        In Most Lysates, Cyclin Destruction Was Followedby
    envelope breakdown, occurs '~,82 min. By ~90 min, the                           at Least One Additional Round of Cyclin Synthesis
    chromosomes become fully aligned on the metaphase plate;                        and Destruction
    anaphase begins shortly thereafter, '~92 minutes, and is fol-                   Most lysates displayed at least one full round of cyclin de-
    lowed by cytokinesis at 95 min. The destruction of cyclin A                     struction and reappearance in vitro (Fig. 3 a), and some went
    begins several minutes before the onset of anaphase and is                      on to destroy the cyclins a second time although with con-
    completed within 5-6 min. Destruction of cyclin B begins a                      siderably slower kinetics (not shown). The reappearance of


    Luca and Ruderman Cyclin Destruction in a Cell-free System                       1897
Published November 1, 1989

                     Meiosis     I          II              Mitosis I        Mitosis 2                     Figure L Timing of the first four
                                                                                                           cell cycles in Spisula embryos. Full-
                                                                                                           grown Spisula oocytes are naturally
                    I                                                                 l                    arrested at the G2/M border of mei-
                                                                                                           osis I with an intact nuclear enve-
            l
     Fertilization
                                   l
                                aria
                                            I
                                           ana                ana
                                                                 I                    I
                                                                                    aria                   lope and a 4C set of partially con-
                                                                                                           densed chromosomes. Fertilization
                                                                                                           activates the resumption of meiosis
                                                                                                           and leads to the mitotic cleavagedi-
                                                                                                           visions. When cultured at 18°C, fer-
            I     I    I     I       I   I      I   I     I    I     I     I     I      I    I             tilized oocytes enter M phase of
           0          20          40           60        80         100        120         140 rnin
                                                                                                           meiosis I at 11-12 min, marked by
                                                                                                           nuclear envelope breakdown and an
     increase in chromosome condensation. The maternal chromosomes go through anaphase of the two meiotic divisions, I and II, at 40 and
     55 min, respectively. After anaphase II, the remaining haploid set of maternal chromosomes and the haploid sperm nucleus decondense
     fully and form two haploid pronuclei; this occurs around 60 minutes and marks the transition into interphase of the first mitotic cleavage
     cycle. Embryos enter M phase of mitosis 1 around 82 min (marked by nuclear envelope breakdown, chromosome condensation), begin
     to exit M phase of mitosis 1 around 92 minutes (marked by the onset of anaphase 1) and pass through the second mitotic cycle as indicated.
     Thin line, interphase; heavy line, M phase; ana, anaphase.



    labeled cyclins in vitro was surprising at first, as we had             in vitro. Furthermore, the protease activity is remarkably
    made no attempt to optimize lysates for protein synthesis.              specific: by the end of a 5-h incubation at 18°C, and in the
    Blocking protein synthesis in M phase lysates did not in any            absence of any exogenous protease inhibitors, only the cy-




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    way affect the timing or extent of cyclin destruction but did           clins were targets for destruction. Even after 24 h the bulk
    prevent the reappearance of cyclin (Fig. 3 b), just as in vivo          of the proteins remained intact (not shown).
    (Hunt, T., and J. Ruderman, unpublished). These results in-                Not all lysates were equally active in protein synthesis.
    dicate that the cyclin proteases and other components respon-           Furthermore, multiple rounds of freezing and thawing re-
    sible for cyclin destruction are present in early M phase cells         sulted in progressive loss of protein synthesis activity. Be-
    at levels sufficient to ensure rapid and complete degradation           cause the major issue addressed here was the regulation of
                                                                            cyclin proteolysis, and ongoing protein synthesis was not
                                                                            needed for cyclin destruction (and, in fact, often partly ob-
                                                                            scured it), we did not attempt to optimize the lysates for pro-
                                                                            tein synthetic activity.

                                                                            Cyclin Destruction In Vitro Does Not Reveal
                                                                            Any Intermediates
                                                                            Once cyclin destruction begins in vivo, it proceeds rapidly
                                                                            and without the appearance of any detectable intermediates
                                                                            (Evans et al., 1983; Swenson et al., 1986; Standart et al.,
                                                                            1987; Westendorf et al., 1989). Because the duration of cy-
                                                                            clin destruction in vitro is slightly longer, it seemed possible
                                                                            that we might be able to visualize transient intermediates by
                                                                            immunoprecipitation. This approach would be particularly
                                                                            useful in detecting a diffuse ladder of higher molecular weight
                                                                            intermediates of the type expected for ATP-dependent, ubi-
                                                                            quitin-mediated proteolysis (reviewed by Rechsteiner, 1987).
                                                                            No such intermediates of cyclin A were apparent in immune
                                                                            precipitates from the cell-free system (Fig. 4), even after long
                                                                            exposures. Immunoblots using the same polyclonal antise-
                                                                            rum, which is directed against >75 % of the length of cyclin
                                                                            A, confirmed this (not shown). Immune precipitates of cyclin
                                                                            B revealed a few faint, more rapidly migrating bands but no
                                                                            larger forms. However, these could represent products of a
                                                                            nonspecific, low level protease present in the antiserum.
    Figure 2. Cyclin destruction in vitro. Embryos were labeled with
    [3SS]methionineduring the first mitotic cell cycle, and a lysate was       These results show that intermediates of cyclin proteolysis
    prepared from early M phase ceils and incubated at 18°C as de-          are destroyed nearly as rapidly in the cell-free system as they
    scribed in the text. After the start of the incubation in vitro (t =    are in the intact cell. They also suggest that the lag in the ki-
    0 min), samples were taken at the indicated times and analysed by       netics of cyclin destruction in vitro is due to deficiencies in
    gel electrophoresis followed by automdiography. The positions of        activities that may mark the cyclins for destruction or to
    cyclin A, cyclin B, and ribonucleotide reductase (RR) are indicated     deficiencies in the activation of cyclin proteases.
    on the left. The positions of molecular weight markers are indicated       Cyclin B occasionally splits into a tight doublet (compare
    by dashes on the right, from top to bottom: 116, 94, 56, and 40 kD.     Figs. 2, 4, 5, and 7 with Fig. 6). In light of other work (Stan-



    The Journal of Cell Biology, Volume 109, 1989                           1898
Published November 1, 1989




                                                                                                 Figure 3. Cyclin destruction is followed
                                                                                                 by resynthesis in vitro. Embryos were
                                                                                                 labeled with [35S]methionine during
                                                                                                 the first mitotic cell cycle and a lysate
                                                                                                 was prepared from early M phase ceils
                                                                                                 and incubated in vitro as described in
                                                                                                 the text. Portions of the lysate were
                                                                                                 then incubated in (a) the absence or (b)
                                                                                                 presence of 100 ~tM emetine. Samples
                                                                                                 were taken at the indicated times after
                                                                                                 the start of the incubation (t = 0 rain)
                                                                                                 and analyzed by gel electrophoresis




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                                                                                                 followed by autoradiography. The po-
                                                                                                 sitions of cyclin A, cyclin B, and ribo-
                                                                                                 nucleotide reductase (RR) are indicated
                                                                                                 on the left.


    dart et al., 1987; Hunt et al., 1988; Draetta et al., 1988;       took '~10-20 min (see Fig. 7 c, for example) and early inter-
    Swenson et al., 1989; Westendorf et al., 1989), we suspect        phase lysates took even longer, •30-60 min (Fig. 5). This
    the upper band is phosphorylated cyclin B. However, neither       result shows that, whatever sets the timing for the switchover
    band was preferentially proteolysed nor was there any obvi-       to cyclin destruction, it is reproduced fairly well in the cell-
    ous shift from one form to the other during the in vitro de-      flee system.
    struction assay.                                                     Spisula embryos, like others, require protein synthesis up
                                                                      to a particular point in early interphase, the "commitment
    B e h a v i o r o f l n t e r p h a s e Lysates                   point, ~ ( ~ 6 5 minutes after fertilization) in order to proceed
    Whereas M phase lysates took 5-15 min to reach the cyclin         into and through the subsequent M phase (Hunt, T., and J.
    destruction point (Figs. 2, 3, and 7 a), mid-interphase lysates   Ruderman, unpublished; reviewed by Swenson et al., 1989).


                                                                                                   Figure 4. Immunoprecipitation of cy-
                                                                                                   clins A and B from [35S]methionine-
                                                                                                   labeled M-phase lysate. Embryos
                                                                                                   were labeled with 35S-methionine dur-
                                                                                                   ing the first mitotic cell cycle, and a
                                                                                                   lysate was prepared from early M
                                                                                                   phase cells and incubated in vitro.
                                                                                                   Samples were taken at the indicated
                                                                                                   times after the start of the incubation
                                                                                                   (t = 0 min) and analyzed by gel elec-
                                                                                                   trophoresis followed by autoradiog-
                                                                                                   raphy. This lysate showed very little
                                                                                                   protein synthesis activity so emetime
                                                                                                   was not added to the incubation mix.
                                                                                                   (a) Total 35S-labeled proteins. (b)
                                                                                                   35S-labeled proteins immunoprecipi-
                                                                                                   rated with polyclonal cyclin A or cy-
                                                                                                   din B antibodies. No 35S-labeled
                                                                                                   proteins immunoprecipitated when
                                                                                                   preimmune sera were used (not
                                                                                                   shown). The positions of molecular
                                                                                                   weight markers are indicated by
                                                                                                   dashes on the right of a, from top to
                                                                                                   bottom: 116, 94, 68, 56, 40, and 21 kD.



     Luca and Ruderman Cyclin Destruction in a Cell-freeSystem         1899
Published November 1, 1989




                                                                                                       Figure 5. The cyclins present in
                                                                                                       early interphase lysates are not
                                                                                                       destroyed in vitro if protein syn-
                                                                                                       thesis is inhibited. Embryos were
                                                                                                       labeled with [35S]methioninedur-
                                                                                                       ing the first mitotic cell cycle and
                                                                                                       a lysate was prepared from early
                                                                                                       interphase cells. Portions of the
                                                                                                       lysate were incubated in the ab-
                                                                                                       sence (control) or presence (eme-
                                                                                                       tine) of 100/~Memetine. Samples
                                                                                                       were taken at the indicated times
                                                                                                       after the start of the incubation (t
                                                                                                       = O min) and analyzed by gel
                                                                                                       electmphoresis followedby auto-
                                                                                                       radiography. The positions of cy-
                                                                                                       clin A, cyclin B, and ribonucleo-
                                                                                                       tide reductase (RR) are indicated.




                                                                                                                                              Downloaded from jcb.rupress.org on May 6, 2011
    These protein synthesis requirements were completely re-           mid interphase lysate, radiolabeled M phase substrate cyclin
    produced in the cell-free system: lysates made from early in-      A was stable for 10-20 min and then began to be destroyed,
    terphase cells taken before the commitment point required          i.e., with typical midinterphase kinetics; cyclin B levels
    further protein synthesis to reach the cyclin destruction point    declined later (Fig. 7 c). This mixed lysate did not require
    (Fig. 5), whereas those from mid interphase (Fig. 7) or M          ongoing protein synthesis to reach the onset of cyclin de-
    phase cells (Figs. 2 and 3) did not. Certain early-interphase      struction (Fig. 7 d). When added to a concentrated early in-
    lysates made around the time of the commitment point did           terphase lysate, the radiolabeled M phase cyclin substrates
    not require protein synthesis to proceed to the cyclin destruc-    were stable for even longer. Cyclin A destruction began after
    tion point, but took a very long time to reach that point (not    60 min; cyclin B destruction began even later and never
    shown). This delay suggests that once the threshold level of       reached completion during the 5 h assayed. Furthermore,
    cyclin is reached, there is an additional rate limiting process    when emetine was added to the dilute M phase-concentrated
    required before various M phase events including cyclin de-       early interphase lysate mix at the beginning of the incuba-
    struction can be activated.                                       tion, the M phase cyclin substrates failed to be destroyed.
                                                                         In all cases examined, the ability of the labeled substrate
    The 7iming of CycUn Destruction Is Set By the Cell                cyclins to undergo destruction, and the timing of their de-
    Cycle Stage of the Lysate, Not the Cell Cycle Stage of            struction, was determined by the concentrated host lysate
    the Cyclins                                                       (summarized in Table I). Thus, the timing of cyclin destruc-
    The kinetics of cyclin destruction in M phase lysates were        tion is set by the cell cycle stage of the lysate, not by the cell
    indistinguishable in concentrated lysates and lysates diluted     cycle stage of the cyclins. If cyclins are in fact marked for de-
    with one volume buffer (not shown). When lysates were             struction at a particular point in the cell cycle by some sort
    diluted further, there were corresponding increases in the        of posttranslational modification, that marking must be re-
    times taken to reach the onset of cyclin destruction (Fig. 6).    versible or subservient to other cues. Furthermore, lysates
    At the highest dilution shown (6.3 %), cyclins were stable for    and intact cells show the same requirements for protein syn-
    >5 h (Fig 6, bottom). Dilution of interphase lysates to 6.3%      thesis to reach the cyclin destruction point. Most impor-
    similarly blocked cyclin destruction (not shown).                 tantly, this requirement extends to substrate cyclins provided
       To see whether the kinetics of destruction are determined      from cells that have progressed well beyond the commitment
    by the cyclins themselves or by other components of the ly-       point for mitosis.
    sates, we asked if radiolabeled %ubstrate" cyclins within a di-      If protein synthesis is blocked past the commitment point,
    lute lysate made at one stage of the cell cycle would be recog-   the cells proceed into and through M phase, destroy the cy-
    nized and destroyed by a concentrated "host" lysate made at       clins on schedule, and arrest in the next interphase. Lysates
    a different stage of the cell cycle. When incubated alone, the    made from these arrested cells were tested for proteolytic ac-
    radiolabeled cyclins in dilute M phase lysates (Fig. 7 a) or      tivity towards exogenous cyclin, which was provided as a di-
    dilute interphase lysates (not shown) were stable. When           lute, radiolabeled M phase lysate. The emetine-arrested in-
    added to a concentrated unlabeled M phase lysate, the radio-      terphase lysates did not contain detectable cyclin proteolytic
    labeled M phase substrate cyclins were destroyed in <10 min       activity at any of the time points of the assay up to 5 h (not
    and, as expected, their destruction did not require ongoing       shown). This result establishes three points. First, it shows
    protein synthesis (Fig. 7 b). When added to a concentrated        that ongoing protein synthesis is not needed to turn off cyclin



   The Journal of Cell Biology, Volume 109, 1989                      1900
Published November 1, 1989


                                                                            early interphase lysate (containing the labeled cyctin to be
                                                                            monitored) was mixed with one volume of concentrated, un-
                                                                            labeled M phase lysate. Emetine was added to the mix to
                                                                            block any new cyclin synthesis that might obscure the disap-
                                                                            pearance of old cyclins. The labeled cyclins (from the early
                                                                            interphase lysate) began to disappear with typical M phase
                                                                            kinetics, i.e., within 10-15 min (Fig. 8 a). When the concen-
                                                                            trated, unlabeled M phase lysate was preincubated alone at
                                                                            18°C for 5 min and then added to one volume of the radiola-
                                                                            beled early interphase lysate, the radiolabeled interphase cy-
                                                                            clins were degraded within 5-10 min, i.e. 5 rain faster (Fig.
                                                                            8 b). These results indicate the M phase lysates are dominant
                                                                            over interphase lysates.

                                                                            lnhibitors and Cyclin Destruction Activity
                                                                            One useful feature of the cell-free system is that it provides
                                                                            the opportunity to test a wide range of protease inhibitors and
                                                                            other reagents that cannot enter intact cells. In most cases,
                                                                            emetine was added to lysates so that ongoing protein synthe-
                                                                            sis would not mask any weak proteolytic events. Most of the
                                                                            inhibitors tested had no effect on either the timing, duration
                                                                            or extent of cyclin destruction (see Table II). Only tosyl-
                                                                            lysine chloromethyl ketone (TLCK) unambiguously inhib-




                                                                                                                                               Downloaded from jcb.rupress.org on May 6, 2011
                                                                            ited destruction (Fig. 9). TLCK is most commonly used as
                                                                            an irreversible inhibitor of trypsin-like serine proteases, but
                                                                            is known to block some cysteine proteases by alkylating sulf-
                                                                            hydryl groups (see Table II for references to TLCK and other
    Figure 6. Small dilutions of concentrated lysates retard cyclin de-     agents tested). Calpain inhibitor I, a cysteine protease inhibi-
    struction; larger dilutions block cyclin destruction altogether. Em-
    bryos were labeled with [3SS]methionineduring the first mitotic         tor specific for calcium-activated calpain-like proteases,
    cell cycle and a concentrated lysate was prepared from early M          retarded cyclin degradation but did not completely block it
    phase cells as described in the text. One aliquot of the concentrated   (Fig. 9). Calpain inhibitor II had no effect. Freshly prepared
    lysate was diluted with buffer to 0.50 of the reaction volume, which    PMSF (up to 2 raM), a general serine protease inhibitor, had
    represented the standard lysate. Successively greater dilutions of      no effect. Other common serine protease inhibitors such as
    the concentrated lysate were made to give reactions in which the        4-amidophenyl-methanesulfonyl fluoride (APMSF), apro-
    lysate represented 0.25, 0.125, or 0.063 of the total incubation vol-   tinin, chymostatin, hemin, and soybean trypsin inhibitor
    ume. Samples were taken from each reaction mix at the indicated         (SBTI) had no effect, ot-2-macroglobulin, which is consid-
    times after the start of the incubation (t --- 0 min) and analyzed by   ered to be a "general" protease inhibitor, did not block cyclin
    gel electrophoresis followedby autoradiography. To compare the ki-
    netics of cyclin destruction in the standard lysate with those in the   destruction in vitro. The general cysteine protease inhibitor
    more dilute lysates, the autoradiograms were exposed for appropri-      E64 (Fig. 9) and its lipid soluble analogue E64d also had no
    ately different times. The positions of cyclin A, cyclin B, and ribo-   effect. However, several agents that interfere with sulfhydryl
    nucleotide reductase (RR) are indicated.                                groups and are commonly used as inhibitors of cysteine pro-
                                                                            teases were very effective in blocking cyclin destruction.
                                                                            These included p-hydroxymercuribenzoic acid (pHMB),
    destruction activity, but is needed for its reappearance. Sec-          n-ethylmaleimide (NEM), ZnCl2 (see below) and CuCI2.
    ond, if cyclins need to be marked by a posttranslational                Iodoacetamide, which also inhibits many cysteine proteases,
    modification in order to enter the cyclin destruction pathway,          had no effect on cyclin destruction. Taken together, these
    then that marking must be reversible and marked M phase                 results are most consistent with the idea that both cyclin A
    eyclins must become unmarked in the emetine-arrested inter-             and cyclin B are destroyed by cysteine proteases, but cer-
    phase lysates. Third, it rules out the idea that cells are main-        tainly do not rule out other explanations such as the involve-
    rained in interphase by a constitutively activated cyclin de-           ment of a novel seriue protease. We note that SBTI, which
    struction pathway.                                                      fails to block cyclin destruction in clam lysates, stabilizes
                                                                            MPF activity after microinjection into pre-meiotic oocytes
    M Phase Lysates are Dominant over lnterphase Lysates                    (Picard et al., 1985) but, because of differences in experi-
    The scheduled cyclin destruction process that operates in in-           mental design, it is difficult to make meaningful compar-
    tact M phase cells, and in lysates made from them, is inde-             isons,
    pendent of ongoing protein synthesis. In contrast, when pro-
    tein synthesis is blocked in early interphase cells taken before        Cyclin Destruction is an ATP-dependent Process and
    the commitment point, and in lysates made from them, cy-                May Require Phosphorylation
    clins fail to be destroyed. We exploited these features to ask          Neither cyclin destruction nor resynthesis was affected by ad-
    if interphase cells contain a dominant inhibitor of cyclin pro-         dition of ATP. When endogenous ATP stores were depleted,
    tease activity. 1 vol of [3sS]methionine labeled, concentrated          cyclin destruction was inhibited almost completely. Cyclin



     Luca and Ruderman Cyclin Destruction in a Cell-free System             1901
Published November 1, 1989




    Figure 7. 3sS-cyclins in diluted M phase lysates are stable, but can serve as substmtes for proteolysis when added to concentrated lysates.
    Embryos were labeled with [35S]methionine during the first mitotic cell cycle. A concentrated lysate was made and diluted 16-fold to give
    a ratio of 0.063 lysate/total volume, as described in the text. The behavior of the radiolabeled cyclins in this dilute lysate (the "substrate"
    cyclins) is shown in a. The behavior of the radiolabeled substrate cyclins from the dilute lysate was then followed in the presence of: (b)
    concentrated M phase lysate and 100 #M emetine; (c) concentrated mid-interphase lysate; (d) concentrated mid-interphase lysate and 100
    #M emetine; (e) concentrated early-interphase lysate; ( f ) concentrated early-interphase lysate and 100/~M emetine. After the start of the
    incubation (t = 0 min), samples were taken at the indicated times and analyzed by gel electrophoresis followed by autoradiography. The




                                                                                                                                                                         Downloaded from jcb.rupress.org on May 6, 2011
    positions of cyclin A, cyclin B, and ribonucleotide reductase are indicated on the right side of e.

    destruction was blocked by EDTA; this was partially re-                              in vitro, and the patterns of phosphorylation are changed
    versed by the subsequent addition of excess Mg ~÷. Addition                          markedly when the in vitro kinase reactions are carried out
    of the kinase inhibitor 600/~M 6-DMAP (6-dimethylamino-                              in the presence of 1 mM ZnCI2 (Reed et al., 1985; Swen-
    purine) strongly inhibited cyclin destruction in vitro (Table                        son et al., 1989; Westendorfet al., 1989). Furthermore, zinc
    II). These results indicate that there is at least one ATP-                          strongly inhibits the activity of histone HI kinase (Pelech et
    dependent step needed for cyclin destruction. Obvious candi-                         al., 1987) which, at least under some circumstances, appears
    dates include ATP-dependent proteolysis, possibly mediated                           to be the same as MPF which, in turn, appears to be a com-
    by ubiquitin, and ATP-dependent phosphorylation (reviewed                            plex of cyclin, p34 co~2asprotein kinase, and a small number
    by Bond and Butler, 1987; Rechsteiner, 1987).                                        of other proteins (reviewed by Featherstone, 1989, but see
                                                                                         also Labb6 et al., 1989). These observations prompted us to
                                                                                         test the effect of zinc on cyclin destruction in vitro. When
    1 mM ZnCl2 Blocks Cyclin Destruction In Vitro                                        1 mM ZnC12 was added to M phase lysates, cyclin destruc-
    Immune precipitates of S. cerevisiae p34 c~c2s, clam cyclin                          tion was inhibited severely (Fig. 10 a). 250/zM ZnC12 was
    A, and clam cyclin B each have substantial kinase activity                           equally effective; 125/~M ZnCI2 delayed destruction by 30-

    Table L Fates of Substrate Cyclins Added to Concentrated Lysates from the Same or Different Stages of the Cell Cycle
    Component I *                          Component 2                         Emetine                              Fate of radiolabeled substrate cyclins
    Conc. M                                    -                                  4-                        Destroyed with M-phase kinetics (5-15 min)
    Dilute M                                   -                                  +                                            Stable
    Dilute M                                Conc. M                               4-                             Destroyed with M-phase kinetics
    Dilute M                             Conc. mid Int                            4-                    Destroyed with mid interphase kinetics (10-20 min)
    Dilute M                            Conc. early Int                           -                     Destroyed with early interphase kinetics (30-60 m/n)
    Dilute M                            Conc. early Int                           +                                            Stable
    Dilute M                          Conc. em. arr. 2-cell                       4-                                           Stable
    Conc.    mid   Int                         -                                  4-                            Destroyed with mid interphase kinetics
    Dilute   mid   Int                         -                                  4-                                           Stable
    Dilute   mid   Int                      Conc. M                               +                               Destroyed with M phase kinetics
    Dilute   mid   Int                   Conc. mid Int                            4-                            Destroyed with mid-interphase kinetics
    Dilute   mid   Int                Conc. em. art. 2-cell                       +                                            Stable
    Conc.    early   Int                        -                                 -                            Destroyed with early interphase kinetics
    Conc.    early   Int                        -                                 +                                            Stable
    Conc.    early   Int                     Conc. M                              +                               Destroyed with M phase kinetics
    Dilute   early   Int                        -                                 4-                                           Stable
    Component 1, which contained radiolabeled substrate cyclins (*), and component 2, concentrated "hosff lysates, were prepared at the indicated cell cycle stages,
    mixed as described in the text, and the fate of the radiolabeled cyclins were followed. Abbreviations: conc., concentrated; early Int, early interphase; era. an'.
    2-ceU, emetine arrested 2-cell; M, M phase; mid Int. mid interphase.




    The Journal of Cell Biology, Volume 109, 1989                                      1902
Published November 1, 1989


                                                                          proteins were completely resistant to this calcium-activated
                                                                          protease activity, as were the bulk of the proteins seen on
                                                                          Coomassie brilliant blue-stained gels. Thus, while calcium
                                                                          did not trigger widespread, nonspecific proteolysis, more
                                                                          than just the cyclins were destroyed. One obvious possibility
                                                                          is that this represents the combined effects of a calcium-
                                                                          activated cyclin protease and a second, calcium-activated
                                                                          protease that recognizes a different, restricted set of sub-
                                                                          strates.
                                                                             To ask if cyclin is indeed destroyed by a calcium-activated
                                                                          cyclin protease (or marked for proteolysis by a calcium-
                                                                          activated event), we added calcium to lysates in which cyclin
                                                                          destruction had been blocked by various inhibitors. When
                                                                          cyclin destruction was blocked by adding 6-DMAP or de-
                                                                          pleting ATP, subsequent addition of 5 mM CaCl2 o v e r c a m e
    Figure 8. M phase lysate is dominant over interphase lysate. Em-
                                                                          Table I1. Effect o f Various Treatments on Cyclin
    bryos were labeled with [35S]methionine during the first mitotic
                                                                          Destruction In Vitro
    cell cycle and a concentrated lysate was prepared from early inter-
    phase cells. (a) One volume of unlabeled M-phase lysate was mixed     lnhibitors of cyclin destruction in vitro
    with one volume of the 35S-labeled interphase lysate, 100 /~M                                                                              References
    emetine was added to prevent the early interphase from proceeding     ATP depletion
    independently to the cyclin destruction point (see text) and, after
                                                                          6-DMAP                                  600/~M                         1, 2, 3
    the start of the incubation (t = 0 min), samples were taken at the
                                                                          EDTA                                     5 mM




                                                                                                                                                              Downloaded from jcb.rupress.org on May 6, 2011
    indicated times. (b) 1 vol of the unlabeled M phase lysate was pre-
                                                                          CuCI2                                    1 mM
    incubated alone at 18°C for 5 minutes. 1 vol of the 35S-labeled
                                                                          ZnC12                                    1 mM
    early interphase lysate and 100 ~M emetine was then added. Time
                                                                          Calpain inhibitor I                    100/~g/ml                         4
    zero indicates the time at which the early interphase and M phase
                                                                          pHMB                                     5 mM                          5, 6
    lysates were mixed together. Samples were taken at the indicated
                                                                          NEM                                      5 mM                           5, 6
    time and analyzed by gel electrophoresis followed by autoradiogra-
                                                                          TLCK                                   300/~g/ml                      7, 8, 9
    phy. The positions of cyclin A, cyclin B, and ribonucleotide reduc-
    tase (RR) are indicated.                                              No effect on cyclin destruction in vitro
                                                                                                                                               References

    60 min (not shown). Zinc inhibition was blocked when 5 mM             ATP                                         500 ~M
    EGTA was added at the same time (Fig. 10 a) and was re-               ATP regen system
                                                                          ATP-3,-S                               10-500 p,M
    versed when EGTA was added later (Fig. 10 b). The kinetics
                                                                          EGTA                                     10 mM
    of cyclin destruction after EGTA "rescue" were delayed only           CaCI2                                 up to 1 mM
    slightly relative to the control.                                     A23187                                    10 #M
                                                                          LiC1                                      1 mM
    Calcium and Cyclin Destruction                                        NaF                                     500 tzM
    The addition of 5 mM EGTA, an effective calcium chelator              c~-2-macroglobulin                     100/~g/ml                        10
    (Caldwell, 1970), had no effect on either the timing or extent        Aprotinin                              200 t~g/ml                       II
                                                                          Calpain inhibitor II                   100 p,g/ml                      4,5
    of cyclin destruction in vitro, suggesting that calcium was not
                                                                          Chymostatin                            100 btg/mi                       12
    involved in cyclin destruction. However, in view of numerous          E64                                    100 p,g/ml                     13, 14
    reports of transient rises of intracellular free calcium around       E64d                                   500 #g/mi                        15
    the time of nuclear envelope breakdown and the metaphase-             Leupeptin                              100/~g/ml                        12
    anaphase transition (e.g., Poenie et al., 1985, 1986; Twigg           Hemin                                   200/~M                        16, 17
    et al., 1988) and the report that microinjection of the cal-          PMSF                                      5 mM                          18
    cium-activated protease calpain advances the onset of both            APMSF                                  100/~g/ml                        19
    M phase and anaphase in tissue culture cells (Schollmeyer,            SBTI                                   250/~g/ml                      20, 21
    1988), we assayed the effect of added calcium in vitro.               fl-mercaptoethanol                        1 mM
       Addition of up to 1 mM CaCl2 had no effect on the onset,           DTT                                       5 mM
                                                                          Iodoacetamide                            5 mM
    duration, or extent of cyclin destruction in vitro, although it
                                                                          Colcemid                                300/zM
    did block further protein synthesis (Fig. 11 a). We point out,        Emetine                                  100 #M
    however, that the absence of a response to 1 mM CaCI2
    could be due to the presence of effective endogenous calcium          The concentrations listed for "inhibitors of cyclin destruction" were the mini-
                                                                          mum ones needed inhibit cyclin destruction in M phase lysates. The concentra-
    chelating systems in the concentrated lysates (see review by          tions listed for reagents having no effect on cyclin destruction were the highest
    Bode, 1981). In contrast, the addition of 5 mM CaC12 re-              ones tested. References: (I) Rebhun et al., 1973; (2) Neant and Guerrier,
    sulted in a very rapid degradation of the cyclins (Fig. 11 b):         1988; (3) Neant et al., 1989; (4) Crawford et al., 1988; (5) Shaw and Green,
                                                                           1981; (6) Bond and Butler, 1987; (7) Shaw et al., 1965; (8) Penn et al. 1976;
    by the first time point taken (2 min), almost all of cyclin A         (9) Kinzel and Konig, 1980; (10) Barter, 1981; (11) Kassel, 1970a; (12)
    and much of cyclin B were gone. This was quickly followed             Umezawa, 1976; (13) Hanada et al., 1978; (14) Sugita et al., 1980; (15) Shoji-
    by the loss of several other prominently labeled proteins, in-        Kasai et al. 1988; (16) Etlinger and Goldberg, 1980; (17) Haas and Rose,
                                                                           1981; (18) James, 1978; (19) Laura et al., 1980; (20) Kassel, 1970b; (21)
    cluding ribonucleotide reductase. Most other radiolabeled             Picard et al., 1985.




    Luca and Ruderman Cyclin Destruction in a Cell-freeSystem             1903
Published November 1, 1989




     Figure9. Effect of protease inhibitors on cyclin destruction in vitro.
     Embryos were labeled with [3~S]methionineduring the first mi-




                                                                                                                                                    Downloaded from jcb.rupress.org on May 6, 2011
     totic cell cycle and a lysate was prepared from early M phase cells.
     Aliquots of this lysate were mixed with 100 ttM E64d, 300/~g/ml
     TLCK, or 100/~g/ml calpain inhibitor I, as described in the text,
     and the fate of the labeled cyclins were analysed by gel electropho-
     resis followed by autoradiography. The positions of cyclin A, cyclin
     B, and ribonucleotide reductase are indicated.



    the inhibition and led to proteolysis of cyclins and the other
    subset of proteins (not shown). Because this calcium-acti-
    vated protease activity was not ATP-dependent, it seems un-
    likely to be the same entity as cyclin protease. (It is, however,
    possible that high calcium levels override the requirement
    for an ATP-dependent step). In contrast, calcium-activated
    protease activities towards both cyclins and non-cyclin pro-
    teins were inhibited by zinc (Fig. 11 c). Thus, the evidence
    on the involvement of calcium is ambiguous.

    Clam Cyclin B Made in Reticulocyte Lysates Is
    Destroyed in the Clam Cell-free System, But Cyclin A                      Figure10. 1 mM ZnC12inhibits cyclin destruction in vitro; this in-
    Is Not                                                                    hibition can be rescued by the subsequent addition of EGTA. Em-
                                                                              bryos were labeled with [3SS]methionineduring the first mitotic
    When SP6 cyclin B translation products made in reticulocyte               cell cycle and a lysate was prepared from early M phase cells. The
    lysate were added to a clam M phase lysate, most cyclin B                 protein synthesis inhibitor emetine was added to prevent new cyclin
    was destroyed on schedule. Sea urchin B-type cyclin reticu-               synthesis from masking the disappearance of prelabeled cyclins.
    locyte translation product was similarly destroyed in a cell-             The fates of the radiolabeled cyclins in the lysate were monitored
    free system from frog eggs (Murray and Kirschner, 1989).                  under the following conditions: (a) Control, lysate alone; + Zinc,
    In contrast, SP6 cyclin A translation products remained sta-              lysate plus 1 mM ZnCI2 added at 0 rain; + Zinc + EGTA, lysate
    ble for >5 h (Fig. 12). Various control experiments showed                plus 1 mM ZnCI2 and 5 mM EGTA, both added at 0 min. (b)
                                                                              Control, lysate alone; + Zinc + EGTA, lysate plus 1 mM ZnCI2
    that the presence of reticulocyte lysate itself, either dilute or
                                                                              (added at 0 min), plus 5 mM EGTA (added at 31 min). The posi-
    concentrated, had no effect on the onset, duration, or extent             tions of cyclin A, cyclin B, and ribonucleotide reductase are indi-
    of destruction of endogenous or exogenous cyclin A or B (not              cated by dots on the left of a and b.
    shown). To test the idea that cyclin A destruction might re-
    quire cyclin B, or some other newly synthesized protein, we               reticulocyte lysates could explain its resistance to proteolysis
    asked about the behavior of the cyclin A present in the trans-            but, at this point, we cannot say how cyclin A made in re-
    lation products programmed by total two-cell RNA. Out of                  ticulocyte lysate differs from that made in vivo or in clam
    all the abundant proteins encoded by two-cell RNA, only cy-               lysates in vitro. Nevertheless, these results add to the rapidly
    clin B was selectively destroyed (Fig. 12). Improper folding              growing body of evidence that the two cyclin types differ in
    or posttranslational modifications of cyclin A produced in                several fundamental ways.



    The Journal of Cell Biology, Volume 109, 1989                             1904
Published November 1, 1989




                                                                                                                                             Downloaded from jcb.rupress.org on May 6, 2011
                                                                                                 Figure H. Effects of calcium on cyclin
                                                                                                 destruction in vitro. Embryos were la-
                                                                                                 beled with [35S]methionine during the
                                                                                                 first mitotic cell cycle and a lysate was
                                                                                                 prepared from early M phase cells. The
                                                                                                 fates of the cyclins during the incuba-
                                                                                                 tions in vitro were monitored as follows.
                                                                                                 (a) Under standard conditions (control)
                                                                                                 and in the presence of an additional
                                                                                                 1 mM CaCI2 (added at 0 min); (b) un-
                                                                                                 der standard conditions (Control) and in
                                                                                                 the presence of an additional 5 mM
                                                                                                 CaCI2 (added at 0 min); (c) 1 mM ZnC12
                                                                                                 (added at 0 min) followed by 5 rnM
                                                                                                 CaCI2 (added at 60 min). The positions
                                                                                                 of cyclin A, cyclin B, and ribonucleotide
                                                                                                 reductase are indicated on a and b. Lane
                                                                                                 M of the control ofb contains the molec-
                                                                                                 ular weight markers; these axe, from top
                                                                                                 to bottom: 116, 94, 56, and 40 kD.


     Discussion                                                         thawing. Just as in intact cells, the in vitro destruction of cy-
                                                                        clin A began first and was followed soon thereafter by the de-
                                                                        struction of cyclin B. Once initiated, cyclin A destruction
     The Cell-free System                                               proceeded rapidly and efficiently. In almost all cases, cyclin
     We have developed a cell-free system from early Spisula em-        A was completely destroyed within a period of 5 min. The
     bryos that reproduces several aspects of the programmed cy-        behavior of cyclin B was more variable. Its destruction often
     clin destruction and reappearance that occurs during each          took longer and the extent of proteolysis was more variable
     cell cycle in vivo. Cyclins in interphase or M phase lysates       among different preparations. No proteolytic intermediates
     were stable for relatively appropriate lengths of time before      were seen for cyclin A; smaller (45-50 kD) breakdown prod-
     undergoing destruction. Cyclin proteolysis did not require         ucts of cyclin B were sometimes apparent in immune precipi-
     ongoing protein synthesis, and proceeded on schedule even          tates but, for technical reasons, it was hard to judge whether
     in lysates that had been frozen and thawed several times.          these arose from specific or nonspecific proteolytic activity.
     Once past the destruction point, cyclins accumulated again.
     Their reappearance was dependent on new protein synthesis,         Setting the 1Imetablefor Cyclin Destruction
     as expected, but the extent of protein synthetic activity varied   In both intact embryonic cells and in the cell-free system, cy-
     among different lysates and was depressed by freezing and          clin levels are controlled by alternating between long periods




     Luca and Ruderman Cyclin Destruction in a Cell-free System          1905
Published November 1, 1989




                                                                                                                                                       Downloaded from jcb.rupress.org on May 6, 2011
     Figure 12. In vitro translated eyclin B but not cyclin A can be used as substrate for the in vitro cyclin destruction assay. (a) Total two-cell
     embryo RNA was translated in a reticulocyte cell-free system in the presence of [3~S]methionineand the labeled products were added to
     an unlabeled, standard M phase lysate. (This lysate was known, from previous experiments, to carry out programmed destruction of exoge-
     nous substrate cyclins within 10--15min.) Emetine was added at 0 min to block further protein synthesis. Samples were taken at the indicated
     times and analyzed by gel electrophoresis followed by autoradiography. (b) SP6-cyclin A or SP6-cyclin B mRNAs were translated in a
     reticulocyte cell-free system in the presence of [35S]methionine. The labeled translation mixes were added to aliquots of the same unla-
     beled M-phase lysate used in part a. Samples were taken at the indicated times and analyzed as above. Lane M of a contains the molecular
     weight markers; these are, from top to bottom: 116, 94, 56, and 40 kD. Dots, from top to bottom: cyclin A, cyclin B, and RR.


    of stability, during which the cyclins accumulate steadily, and              Second, if protein synthesis is blocked before a certain
    brief bursts of proteolysis, during which the cyclins disap-              point in early interphase, clam embryos fail to enter mitosis
    pear over a period of just a few minutes. How is the oscilla-             and fail to degrade the cyclins. If protein synthesis is blocked
    tion between these two states controlled and what regulates               after this point, the "commitment point," cells will proceed
    the timing of the switches? Several lines of evidence show                through mitosis and degrade the cyclins on schedule (Hunt,
    that (a) cells must build up a certain level ofcyclin to proceed          T., and J. Ruderman, unpublished, reviewed in Swenson et
    at a later time into M phase (b) entry into M phase is a pre-             al., 1989). This protein synthesis requirement is reproduced
    requisite for proceeding to the cyclin destruction point near             in the cell-free system described here. Lysates made from
    the end of M phase.                                                       cells taken before the commitment point required further
       First, introducing cyclin into G2-arrested frog oocytes                protein synthesis to proceed to the cyclin destruction point,
    drives them into M phase in a dose-dependent manner                       whereas lysates made after the commitment point went on
    (Swenson et al., 1986; Pines and Hunt, 1987; Westendorf et                to destroy the cyclins. Furthermore, lysates from emetine-
    al., 1989) and translation of endogenous frog oocyte cyclin               arrested interphase cells never went on to develop cyclin de-
    mRNAs is essential for entry into mitosis (Minshull et al.,               struction activity. Perhaps the most convincing evidence that
     1989). Using an mRNA-dependent cell-free system from                     entry into M phase is required for the subsequent destruction
    frog eggs, Murray and Kirschner (1989) have shown that cy-                of the cyclins comes from the recent work of Murray and
    clin is the only newly synthesized protein needed to drive                Kirschner (1989), who found that low amounts of cyclin both
    nuclei into M phase. In apparent contrast with this idea, Leh-            failed to drive nuclei into M phase and failed to be destroyed.
    ner and O'Farrell (1989) found that mutant Drosophila em-                    Concentrated lysates destroyed cyclins on schedule. Suc-
    bryos with lower cyclin A levels progressed through the later             cessive dilutions led to successive delays in reaching the cy-
    cell cycles with wild-type kinetics and concluded that cyclin             clin destruction point, as would be expected in a simple bi-
    is not the rate limiting component. It is important to point              molecular reaction involving a substrate and its protease. At
    out, however, they did not consider contributions by cyclin               high dilutions, cyclins were stable for many hours. This fea-
    B, also present in Drosophila embryos (Whitfield et al.,                  ture provided the opportunity to use the radiolabeled cyclins
     1989) and that cyclin B can substitute for cyclin A as an in-            present in these very dilute lysates as substrates in other reac-
    ducer of M phase, at least under some circumstances (West-                tions. When substrate cyclins from one stage of the cell cycle
    endorf et al., 1989).                                                     were mixed with concentrated lysates made at a different



    The Journal of Cell Biology, Volume 109, 1989                             1906
Published November 1, 1989


    stage of the cell cycle, the timing of the destruction of the ra-    tion but did not completely block it. We were not able to as-
    diolabeled substrate cyclins was always determined by the            say calpain itself. As a more direct test of the idea that cal-
    cell cycle stage of the concentrated "host" lysate, not by the       cium is a physiological trigger of cyclin destruction, we
    cell cycle stage of the substrate cyclins. These results further     looked at the effect of depleting calcium or adding extra cal-
    show that even if posttranslational modifications such as            cium to the cell-free system. High concentrations of EGTA
    phosphorylation (see below) are used to tag cyclins for subse-       had no effect on timing of the onset, duration or extent of cy-
    quent destruction, this tagging is either readily reversible or      clin proteolysis, and adding up to 1 mM CaCI2 failed to
    is, by itself, insufficient to trigger cyclin destruction.           trigger or accelerate cyclin destruction. However, the addi-
       When equal volumes of two different concentrated lysates,         tion of even higher levels of CaCI2 (5 mM) resulted in the
    one from early interphase and the other from M phase, were           breakdown of a small set of proteins which included the cy-
    mixed together, cyclin destruction proceeded with M phase            clins and ribonucleotide reductase. Calcium-activated cyclin
    kinetics, rather than with kinetics intermediate between early       breakdown was rapid and complete; loss of reductase and a
    interphase and M phase. The outcome of this simple experi-           few other proteins proceeded more slowly (see Fig. 11 b).
    ment reveals two new pieces of information. First, inter-            Although the consequence of calcium addition is obvious
    phase lysates do not contain a dominant inhibitor of cyclin          and dramatic, the interpretation is not. Various approaches
    protease. Second, even though cyclin levels control the tim-         failed to indicate whether this calcium-activated proteolysis
    ing of entry into M phase, and entry into M phase is required        was artifactual or represented the combined effect of two
    for subsequent cyclin destruction, cyclin levels themselves          different calcium-activated pathways, one for cyclins and one
    do not determine when destruction occurs. If cells did initi-        for second restricted set of substrates. More information, es-
    ate cyclin destruction upon reaching a critical cyclin concen-       pecially on the rate of calcium chelation by EGTA and the
    tration, then the mixed lysate with intermediate cyclin levels       effectiveness of calcium sequestering systems in these very
    would have required further cyclin protein synthesis to reach        concentrated lysates, will be needed to sort this out.
    the cyclin destruction point. They do not. Thus we can con-




                                                                                                                                            Downloaded from jcb.rupress.org on May 6, 2011
    clude that, once in M phase, the timing of cyclin destruction        The Cyclin Destruction Pathway
    is set by something other than accumulation of cyclin.               Is periodic cyclin destruction controlled by periodic activa-
        A particularly intriguing question concerns the sequential       tion of cyclin proteases or by period changes in the accessi-
     proteolysis of the two different cyclins. Why is cyclin A de-       bility of the substrate cyclins themselves? Soon after we be-
     stroyed first, then cyclin B? One obvious possibility is that       gan this work, it became obvious that it would be difficult
     there are two different, sequentially activated proteases. Ev-      to answer this question without knowing something about cy-
    ery one of the protease inhibitors and other agents tested           clin protease. Hoping to learn which class of protease is
    affected the destruction of cyclin A and cyclin B equally. This      involved, we tested a wide variety of protease inhibitors in
     indicates that, if there are two different cyclin proteases, they   the cell free system. Only one of these, TLCK, completely
    are remarkably similar and probably arose from a common              blocked cyclin destruction. TLCK, a chloromethyl ketone
     ancestor. The converse explanation is, of course, that both         derivative of lysine, irreversibly inhibits trypsin and trypsin-
    cyclins are degraded by a single protease, and that the timing       like serine proteases by alkylating the histidine residue in the
    of degradation is controlled at the level of substrate accessi-      active site (Shaw et al., 1965) TLCK is not, however, a per-
     bility. At this point, we cannot distinguish between these two      fect diagnostic tool as it can also alkylate thiol groups and
    alternatives.                                                        block the activity of certain cysteine proteases (e.g. Chou et
                                                                         al., 1974; Arnon, 1970) and some kinases are inactivated by
    Calcium and Cyclin Destruction In Vitro                              TLCK alkylation of their sulfhydryl groups (Kinzel and Ko-
    Sparked by Weisenberg's (1972) observation that calcium can          nig, 1980). TLCK thus could be blocking one or more of
    depolymerize microtubules, Kiehart's (1981) demonstration            several different possible steps in the cyclin destruction path-
    that microinjection calcium can dissociate mitotic spindles          way, such as phosphorylation or conformation changes in the
    and numerous indications of intracellular calcium bursts at          cyclins themselves, p34 "~c2/2gprotein kinase, and other cy-
    key morphological transitions during the cell cycle, includ-         clin-associated proteins, and cyclin protease itself, as well as
    ing nuclear envelope breakdown, entry into mitosis, and              directly inhibiting the active site of cyclin protease.
    anaphase onset (e.g. Keith et al., 1985; Hepler, 1985; Poenie           Penn et al. (1976) found that adding a low concentration
    et al., 1985; 1986; Steinhardt and Alderton, 1988; Twigg et          of TLCK to cultures of sea urchin embryos delayed the cell
    al., 1988), there has been persistent enthusiasm for the idea        cycle in an interesting way: cells passed through interphase
    that these calcium bursts actually drive transitions from one        and entered M phase on schedule, but anaphase onset was
    stage of the cell cycle to the next. Calcium sequestering vesi-      delayed considerably. We argued that TLCK was acting as
    cles associated with the mitotic spindle (Silver et al., 1980)       a specific inhibitor of a protease involved in the maintenance
    could supply the calcium for triggering anaphase, although           of cell cycle times rather than a general inhibitor of macro-
    it should be pointed out that there are several conflicting ob-      molecular synthesis. A closer inspection of the delay in clam
    servations on the temporal order of the calcium burst and            embryos shows that TLCK arrests cells in late metaphase
    anaphase onset. Schollmeyer (1988) has reported that injec-          (Luca, F., and J. Ruderman, unpublished). Taken with the
    tions of calpain, a calcium-activated cysteine protease, ac-         demonstration in this paper that TLCK inhibits cyclin de-
    celerates passage into and through mitosis, but certain tech-        struction, we make two conclusions. First, maintaining high
    nical aspects of those experiments preclude a firm conclusion        cyclin levels keeps ceils in M phase. Second, cyclin destruc-
    on this point (see Lee, 1989, for discussion).                       tion, which temporally precedes anaphase onset, is probably
       We found that calpain inhibitor 1, an inhibitor of certain        a requirement for it.
    calcium-activated cysteine proteases, slowed cyclin destruc-            Like TLCK, the ATP requirement for cyclin destruction



    Luca and Ruderman Cyclin Destruction in a Cell-free System           1907
Published November 1, 1989


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     Luca and Ruderman Cyclin Destruction in a Cell-free System                            1909

				
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